Peptidyl transferase substrate activity and inhibition of protein biosynthesis by a hydrophilic-aminoacyl analogue of puromycin.

A puromycin analogue possessing a hydrophilic amino acid, 3′-N-[S-(6-hydroxyhexyl)-$\text{L}$-cysteinyl]puromycin aminonucleoside, has been prepared and examined as a substrate for ribosomal peptidyl transferase. Kinetic studies indicate that this non-aromatic aminoacyl analogue is 95.6% as efficient as the parent antibiotic in the transpeptidation reaction. In addition, the analogue is an effective inhibitor of poly (U) and poly (U,C) directed protein synthesis in an $\text{Escherichia coli}$ cell free system.     

Cloning of a human tRNA isopentenyl transferase

A cDNA of human origin is shown to encode a tRNA isopentenyl transferase (E.C. 2.5.1.8). Expression of the gene in a Saccharomyces cerevisiae mutant lacking the endogenous tRNA isopentenyl transferase MOD5 resulted in functional complementation and reintroduction of isopentenyladenosine into tRNA. The deduced amino acid sequence contains a number of regions conserved in known tRNA isopentenyl transferases. The similarity to the S. cerevisiae MOD5 protein is 53%, and to the Escherichia coli MiaA protein 47%. The human sequence was found to contain a single C2H2 Zn-finger-like motif, which was detected also in the MOD5 protein, and several putative tRNA transferases located by BLAST searches, but not in prokaryotic homologues.     

Peptidyl transferase of bacterial ribosome: resistance to proteinase K.

70-S ribosomes and 50-S ribosomal subunits from Escherichia coli D10 were treated with proteinase K for increasing periods of time. Peptidyl transferase activity and sparsomycin-induced binding of (U)C-A-C-C-A-[3H]Leu-Ac were tested in the treated particles, the binding of the substrate being more sensitive to the protease than peptide bond formation. Comparison of the amounts of proteins present in the treated particles with the residual activity indicates that only proteins L3 and L14 are released at a similar rate to that at which peptidyl transferase activity is lost. Proteins related to this ribosomal activity by other techniques are lost at a faster rate than the activity itself. In addition, the results indicate that sparsomycin stimulates the binding of the substrate by a different mechanism from that which inhibits peptide bond formation.     

Peptidyl transferase centre of bacterial ribosomes: substrate specificity and binding sites.

A detailed scheme of the Peptidyl Transferase Centre of bacterial ribosomes is proposed by summarizing the literature data on the substrate specificity of the acceptor and donor sites. According to the proposed scheme only the elements of the donor and acceptor having a stable structure bind with the ribosome. The present paper proposes such main elements for the donor and acceptor.     

Peptidyl transferase antibiotics perturb the relative positioning of the 3'-terminal adenosine of P/P'-site-bound tRNA and 23S rRNA in the ribosome.

A range of antibiotic inhibitors that act within the peptidyl transferase center of the ribosome were examined for their capacity to perturb the relative positioning of the 3' end of P/P'-site-bound tRNA and the Escherichia coli ribosome. The 3'-terminal adenosines of deacylated tRNA and N-Ac-Phe-tRNA were derivatized at the 2 position with an azido group and the tRNAs were cross-linked to the ribosome on irradiation with ultraviolet light at 365 nm. The cross-links were localized on the rRNA within extended versions of three previously characterized 23S rRNA fragments F1', F2', and F4' at nucleotides C2601/A2602, U2584/U2585 (F1'), U2506 (F2'), and A2062/C2063 (F4'). Each of these nucleotides lies within the peptidyl transferase loop region of the 23S rRNA. Cross-links were also formed with ribosomal proteins L27 (strong) and L33 (weak), as shown earlier. The antibiotics sparsomycin, chloramphenicol, the streptogramins pristinamycin IA and IIA, gougerotin, lincomycin, and spiramycin were tested for their capacity to alter the identities or yields of each of the cross-links. Although no new cross-links were detected, each of the drugs produced major changes in cross-linking yields, mainly decreases, at one or more rRNA sites but, with the exception of chloramphenicol, did not affect cross-linking to the ribosomal proteins. Moreover, the effects were closely similar for both deacylated and N-Ac-Phe-tRNAs, indicating that the drugs selectively perturb the 3' terminus of the tRNA. The strongest decreases in the rRNA cross-links were observed with pristinamycin IIA and chloramphenicol, which correlates with their both producing complex chemical footprints on 23S rRNA within E. coli ribosomes. Furthermore, gougerotin and pristinamycin IA strongly increased the yields of fragments F2' (U2506) and F4' (U2062/C2063), respectively. The results obtained with an RNAse H approach correlate well with primer extension data implying that cross-linking occurs primarily to the bases. Both sets of data are also consistent with the results of earlier rRNA footprinting experiments on antibiotic-ribosome complexes. It is concluded that the antibiotics perturb the relative positioning of the 3' end of the P/P'-site-bound tRNA and the peptidyl transferase loop region of 23S rRNA.     

Metabolism and relative carcinogenic potency of chloroethanes: a quantum chemical structure-activity study

Using the all-valence electron, semiempirical molecular orbital method, MNDO, properties have been identified and calculated for eight chloroethanes which can serve as indicators of their extent of transformation to alcohols by cytochrome P450 and the subsequent formation of aldehydes by loss of HCl from these alcohols. The assumption was made that these aldehydes are the active carcinogens of the chloroethanes and that they act as electrophiles in adduct formation with DNA bases. Electrophilic properties of these putative ultimate carcinogens have been calculated which are indicators of the rank order of carcinogenic activity of the parent compounds in susceptible species. Particularly relevant in this respect are (a) the electron affinity of aldehydes as measured by the energy of their electron accepting (lowest energy virtual) orbital, and (b) the net charge on the C alpha carbon, adjacent to the carbonyl carbon, which can participate in electrophilic attack on nucleophilic sites of DNA bases. The molecular properties identified in this study as indicators of rank order or carcinogenic activity of the parent chloroethanes are consistent with the importance of cytochrome P450 in transforming halohydrocarbons to active carcinogens and of acylchlorides and chloroaldehydes as the active form. Their validity and usefulness can be further tested in screening unknown and more complex chlorohydrocarbons for carcinogenic activity.     

A combined experimental and quantum chemical study on the putative protonophoric activity of thiocyanate

Inhibition of gastric acid secretion by thiocyanate is explained by a protonophoric mechanism assuming that thiocyanate induces a H(+) back flux from the acidic gastric lumen into the parietal cells of gastric mucosa. Protonophoric activity of thiocyanate was examined by swelling measurements using rat liver mitochondria and theoretically by quantum chemical methods. Mitochondria suspended in K-thiocyanate medium plus nigericin (an H/K-exchanger) swelled when the medium pH was acidic, indicating that SCN(-) initiates a transfer of H(+) across the inner membrane. To rationalize the protonophoric activity of thiocyanate, we considered the dehydration of SCN(-) to be critical for transmembranal H(+) transfer. For modeling this process, various hydrate clusters of SCN(-) and Cl(-) were generated and optimized by density functional theory (DFT) at the B3-LYP/6-311++G(d,p) level. The cluster hydration energy was lower for SCN(-) than for Cl(-). The total Gibbs free energies of hydration of the ions were estimated by a hybrid supermolecule-continuum approach based on DFT. The calculated hydration energies also led to the conclusion that SCN(-) is less efficiently solvated than Cl(-). Due to the easier removal of the hydration shell of SCN(-) relative to Cl(-), SCN(-) is favored in going across the membrane, giving rise to the protonophoric activity.     

Evidence for metalloenzyme character of tRNA nucleotidyl transferase.

Previous studies (1–5) have shown that several nucleotidyl transferases are metalloenzymes and in a few cases (1–3) the metal has been identified as zinc. In all instances these enzymes are specifically inhibited by incubation with the chelating agent 1,10-phenanthroline but are not affected by the structurally similar 1,7-phenanthroline which does not chelate metals. We report here that tRNA nucleotidyl transferase from E. coli is inhibited by 1,10-phenanthroline and that only the initial rate of incorporation of AMP is affected; CMP incorporation is not specifically inhibited by this chelator. This finding is in conflict with a previous study (5) in which it was claimed that tRNA nucleotidyl transferase from Rous sarcoma virus and from yeast was unaffected by 1,10-phenanthroline and suggests that the E. coli tRNA nucleotidyl transferase is a metalloenzyme.     

Glucosyl transferase activity of bovine galactosyl transferase

Bovine galactosyl transferase was found to utilize UDPglucose as a substrate and elicit disaccharide biosynthesis with glucose and N-acetylglucosamine as acceptors. The relative rate of glycosyl transferase with N-acetylglucosamine as acceptor was 0.3%, the rate for N-acetyllactosamine biosynthesis. This activity was also evidenced indirectly from NMR water proton relaxation experiments, and from Mn(II) ESR experiments. In direct experiments with radioactive UDPglucose, paper chromatography showed a product which migrated with cellobiose when glucose was the acceptor and a new, glucose-containing product which resulted when GlcNAc was the acceptor.Despite this marginally expanded specificity of the donor site, spin-label experiments with a covalently bound UDPgalactose analog reaffirmed the restrictive nature of the donor site against this non-glycosyl-like analog.     

tRNA structure from a graph and quantum theoretical perspective

One of the objectives of theoretical biochemistry is to find a suitable representation of molecules allowing us to encode what we know about their structures, interactions and reactivity. Particularly, tRNA structure is involved in some processes like aminoacylation and genetic code translation, and for this reason these molecules represent a biochemical object of the utmost importance requiring characterization. We propose here two fundamental aspects for characterizing and modeling them. The first takes into consideration the connectivity patterns, i.e. the set of linkages between atoms or molecular fragments (a key tool for this purpose is the use of graph theory), and the second one requires the knowledge of some properties related to the interactions taking place within the molecule, at least in an approximate way, and perhaps of its reactivity in certain means. We used quantum mechanics to achieve this goal; specifically, we have used partial charges as a manifestation of the reply to structural changes. These charges were appropriately modified to be used as weighted factors for elements constituting the molecular graph. This new graph-tRNA context allow us to detect some structure-function relationships.     

The rational of catalytic activity of herpes simplex virus thymidine kinase. a combined biochemical and quantum chemical study

Most antiherpes therapies exploit the large substrate acceptance of herpes simplex virus type 1 thymidine kinase (TK(HSV1)) relative to the human isoenzyme. The enzyme selectively phosphorylates nucleoside analogs that can either inhibit viral DNA polymerase or cause toxic effects when incorporated into viral DNA. To relate structural properties of TK(HSV1) ligands to their chemical reactivity we have carried out ab initio quantum chemistry calculations within the density functional theory framework in combination with biochemical studies. Calculations have focused on a set of ligands carrying a representative set of the large spectrum of sugar-mimicking moieties and for which structural information of the TK(HSV1)-ligand complex is available. The k(cat) values of these ligands have been measured under the same experimental conditions using an UV spectrophotometric assay. The calculations point to the crucial role of electric dipole moment of ligands and its interaction with the negatively charged residue Glu(225). A striking correlation is found between the energetics associated with this interaction and the k(cat) values measured under homogeneous conditions. This finding uncovers a fundamental aspect of the mechanism governing substrate diversity and catalytic turnover and thus represents a significant step toward the rational design of novel and powerful prodrugs for antiviral and TK(HSV1)-linked suicide gene therapies.